SUMMARY Regulation of transcription by RNA polymerase II (pol II) is essential to control cell growth, differentiation and development. Among the factors required for genome-wide pol II transcription is the evolutionarily conserved, 10-subunit TFIIH complex, which plays fundamental roles in multiple steps of the transcription process including promoter opening and mRNA processing; however, the molecular mechanisms remain unclear. The long term goal of this project is to elucidate the molecular mechanisms by which TFIIH regulates pol II transcription. The rationale for this project is that understanding these mechanisms will reveal the molecular bases for multiple and diverse developmental and somatic diseases, including neurological disorders, progeroid syndromes, skin diseases and cancer. The objectives of this collaborative project are to establish a structural and mechanistic understanding of how TFIIH controls basic transcriptional events such as promoter opening, pol II pausing, and mRNA processing. We recently completed structural analysis of native TFIIH complexes (both human and yeast) using chemical crosslinking/mass spectrometry (CXMS) and 3D modeling. The results predict two distinct, conserved protein-protein interaction networks involving the essential enzymatic subunits XPB/Ssl2 and CDK7/Kin28. The ATPase/translocase XPB/Ssl2 is required for transcription initiation, as it melts the promoter DNA template to allow single-stranded DNA to engage the pol II active site. The CDK7/Kin28 kinase phosphorylates the C-terminal domain (CTD) of the pol II large subunit as well as other key substrates (e.g. P-TEFb) that collectively enable precise regulation of pol II initiation, pausing, and mRNA processing. In Aim 1 we will investigate how the XPB/Ssl2 ATPase/translocase is regulated by an interaction network involving p52/Tfb2 and p8/Tfb5; in Aim 2 we will investigate how specific residues and domains within CCNH/Ccl1 and MAT1/Tfb3 govern CDK7/Kin28 kinase activity. To accomplish these goals, we will utilize a powerful combination of yeast genetics, site-directed insertion of UV-responsive crosslinkers, and in vivo functional screens to structurally refine and functionally validate protein-protein interfaces within TFIIH. These data will guide a series of in vitro and cell-based assays in human cells that will rigorously test the roles of specific molecular interfaces in the regulation of TFIIH function in transcription, genome-wide. We will employ methods that are well-established by the PIs and our collaborators, including reconstituted in vitro transcription, permanganate footprinting (in vitro and in cells), GRO-Seq, ChIP or ChIP-Seq, and RNA-Seq. If successful, these experiments will allow us to link a given protein-protein interface to regulation of specific transcription-associated events such as pol II pausing, promoter opening, or alternate splicing. Finally, potent and specific chemical inhibitors of XPB and CDK7 will be evaluated to more clearly define their roles in general and gene-specific (p53 response will be studied here) transcription while serving as key positive controls for disruption of XPB or CDK7 activity.